ESTs are sequences derived from randomly selected clones from various cDNA libraries (Adams, M. D. et al. Science 252:1651-1656 (1991); Adams, M. D. et al. Nature 377:3-174 (1995); Adams, M. D. et al., Nature 355:632-634 (1992); Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997)). Each cDNA clone is generated from a transcript, and the frequency and distribution of the many different transcripts in any given tissue depends on the tissue specific activity of the genes. The translation of transcript frequency and distribution into frequency and distribution of EST sequences depends not only on the specificity and magnitude of mRNA expression, but also on other factors such as mRNA stability and clonability of these EST sequences. Therefore, a specificity or frequency analysis of ESTs only provides a guide for the prediction of expression patterns. Nevertheless, ESTs provide a valuable source of information that may be utilized to predict the expression patterns of specific genes in different tissues.
The recently developed NCI Cancer Genome Anatomy Project (CGAP) uses microdissection and laser-capture techniques to generate defined and tissue/tumor specific EST libraries (http://www.ncbi.nlm.nih.gov/ncicgap (Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997)). CGAP has already accumulated a vast number of tissue-specific sequences and the CGAP sequence data base is rapidly growing with the continuous addition of sequences from different tissues and tumor types. There are many ways by which the EST sequence data can be processed to cluster, sort and filter the cDNA sequences, in order to identify genes that are specifically expressed in certain tissues. Database “mining” for cDNAs that are preferentially or exclusively expressed in defined tissues, or in malignant/neoplastic tissues provides lists of potential target genes for cancer therapy (Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997); Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304 (1998)). Although in many cases these “candidate genes”, which appear tissue specific in database analyses, cannot be confirmed in their specificity by experimental techniques (e.g. Northern blots or PCR), a reasonable number of candidate genes remain for which the predicted and desired expression pattern can be experimentally confirmed (Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304 (1998); He, W. W. et al., Genomics 43:69-77 (1997)). These specifically expressed genes are of interest because of their functions in cell or tumor biology and may also be directly used as markers for cancer diagnosis and as the basis for a variety of methods of therapy.
One method of therapy is to use the gene product as a vaccine to enhance the patient's immune response to the cancer. T cells play an important role in tumor regression in most murine tumor models. Tumor infiltrating lymphocytes (“TIL”) that recognize unique cancer antigens can be isolated from many murine tumors. The adoptive transfer of these TIL plus interleukin-2 can mediate the regression of established lung and liver metastases (Rosenberg, S., et al., Science 233:1318-1321 (1986). In addition, the secretion of IFN-.gamma. by injected TIL significantly correlates with in vivo regression of murine tumors suggesting activation of T-cells by the tumor antigens. (Barth, R., et al., J. Exp. Med. 173:647-658 (1991)). In humans, the ability of tumor TIL to mediate the regression of metastatic cancer in 35 to 40% of melanoma patients when adoptively transferred into patients with metastatic melanoma attests to the clinical importance of the antigens recognized (Rosenberg, S., et al., N Engl J Med 319:1676-1680 (1988); Rosenberg S., J. Clin. Oncol. 10:180-199 (1992)).
T cell receptors on CD8+ T cells recognize a complex consisting of an antigenic peptide (9-10 amino acids for Human Leukocyte Antigen (“HLA”)-A2), β-2 microglobulin, and class I major histocompatibility complex (“MHC”) heavy chain (HLA-A, B, C, in humans). Peptides generated by digestion of endogenously synthesized proteins are transported into the endoplasmic reticulum, bound to class I MHC heavy chain and β-2 microglobulin, and finally expressed in the cell surface in the groove of the class I MHC molecule. Therefore, T cells can detect molecules that originate from proteins inside cells, in contrast to antibodies that detect molecules expressed on the cell surface. Antigens recognized by T cells thus may be particularly useful for inhibiting the progression of cancer.
Strong evidence that an immune response to cancer exists in humans have been provided by, for example, the existence of lymphocytes within melanoma deposits. These lymphocytes, when isolated, are capable of recognizing specific tumor antigens on autologous and allogeneic melanomas in an MHC restricted fashion. (Kawakami, Y., et al., J. Immunol. 148:638-643 (1992); Hom, S., et al., J. Immunother. 13:18-30 (1993)). TIL from patients with metastatic melanoma recognize shared antigens including melanocyte-melanoma lineage specific tissue antigens in vitro (Kawakami, Y., et al., J. Immunother. 14:88-93 (1993); Anichini, A. et al., J. Exp. Med. 177:989-998 (1993)).
Although several tumor associated antigens (“TAA”) have been found for melanoma, there is a need to identify tissue specific genes whose expression is associated with cancers of other tissues.